Prevalence and Clinicopathological Significance of MET Overexpression and Gene Amplification in Patients with Gallbladder Carcinoma

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Open Access

Prevalence and Clinicopathological Significance of MET Overexpression and Gene Amplification in Patients with Gallbladder Carcinoma

Original Article

Purpose

Mesenchymal epithelial transition (MET) is a proto-oncogene that encodes a heterodimeric transmembrane receptor tyrosine kinase for the hepatocyte growth factor. Aberrant MET signaling has been described in several solid tumors—especially non-small cell lung cancer—and is associated with tumor progression and adverse prognosis. As MET is a potential therapeutic target, information regarding its prevalence and clinicopathological relevance is crucial.

Materials and Methods

We investigated MET expression and gene amplification in 113 gallbladder cancers using tissue microarray. Immunohistochemistry was used to evaluate MET overexpression, and silver/fluorescence in situhybridization (ISH) was used to assess gene copy number.

Results

MET overexpression was found in 37 cases of gallbladder carcinoma (39.8%), and gene amplification was present in 17 cases (18.3%). MET protein expression did not correlate with METamplification. METamplification was significantly associated with aggressive clinicopathological features, including high histological grade, advanced pT category, lymph node metastasis, and advanced American Joint Committee on Cancer stage. There was no significant correlation between any clinicopathological factors and MET overexpression. No difference in survival was found with respect to MET overexpression and amplification status.

Conclusion

Our data suggested that MET might be a potential therapeutic target for targeted therapy in gallbladder cancer, because METamplification was found in a subset of tumors associated with adverse prognostic factors. Detection of METamplification by ISH might be a use- ful predictive biomarker test for anti-MET therapy.

Key words

Gallbladder cancer, Gene amplification, Gene copy number, In situhybridization, Immunohistochemistry, MET

Yeseul Kim, MD, PhD Seong Sik Bang, MD Seungyun Jee, MD Sungeon Park, MD Su-Jin Shin, MD, PhD Kiseok Jang, MD, PhD

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Correspondence: Kiseok Jang, MD, PhD Department of Pathology, Hanyang University College of Medicine, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Korea Tel: 82-2-2290-8248

Fax: 82-2-2296-7502

E-mail: [email protected] Received July 2, 2019

Accepted October 23, 2019 Published Online October 24, 2019 Department of Pathology, Hanyang University College of Medicine, Seoul, Korea

Introduction

Gallbladder cancer (GBC) is a relatively rare primary malig- nancy, but it is the most common malignant neoplasm of the biliary tract, and one of the most aggressive cancers of the biliary tract with poor prognosis [1]. A variety of genetic and epigenetic alterations are associated with GBC, involving tumor suppressor genes, oncogenes, and DNA repair genes.

Previous studies have focused on the importance of oncogenes (epidermal growth factor receptor [EGFR], K-Ras, and ERBB2), tumor suppressor genes (p53), cell cycle regulators (cyclin D1, cyclin E), and micro-RNAs in the development and prognosis of GBC. However, the precise information regarding genetic and molecular alterations in GBC is still poorly understood [2]. Li et al. [3] investigated genetic alterations in 57 GBC cases by whole exome and targeted sequencing, and found frequent mutations of TP53, KRAS, and

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ERBB3. Moreover, ErbB signaling, including EGFR, ERBB2, ERBB3, ERBB4, and their downstream genes is the most extensively mutated pathway, affecting 36.8% of patients with GBC. Although cytotoxic chemotherapy with the combination of gemcitabine and cisplatin has been accepted as the first-line therapy, the patient outcome is still disappoint- ing. After ErbB signaling pathway was found to be one of the pathways contributing to gallbladder carcinoma, target-ori- ented agents, including monoclonal antibodies and tyrosine kinase inhibitors against EGFR and vascular endothelial growth factor have entered clinical trials [4,5]. However, as those clinical trials revealed no, or marginal benefits, novels targets are needed to improve the patient outcomes.

The mesenchymal epithelial transition (MET) receptor tyrosine kinase pathway is one of the most commonly activated signaling pathways in primary human cancers. MET has a heterodimeric structure composed of an extracellular

!subunit and a "chain that contains an extracellular domain, a transmembrane segment, and an intracellular tyrosine kinase region [6]. Activation of MET signaling has a critical role not only in tumor formation, progression, angiogenesis, and metastasis, but is also involved in mediating resistance against other targeted treatments [7,8]. Hepatocyte growth factor (HGF), a ligand of MET, stimulates the tyrosine kinase activity of MET and activates multiple downstream signaling pathways, including the Grb2-Ras–mitogen-activated protein kinase (MAPK) cascade, the phosphatidylinositol-3 kinase (PI3K) pathway, the Wnt/"-catenin pathway, and the signal transducer and activator of transcription (STAT) pathway [9]. Dysregulation of MET protein has been reported in a variety of human malignancies and premalignant lesions, including hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC) [10,11]. Accordingly, the possibility of targeting the METgene in different cancer types is now being evaluated in clinical trials. Several drugs have been developed that target MET or HGF. These agents are divided into small molecule inhibitors and monoclonal antibodies.

The small molecule tyrosine kinase inhibitors are further sub- divided into multiple kinases and selective MET inhibitors.

Crizotinib and cabozantinib are examples of multikinase MET inhibitors. Selective MET inhibitors include capmatinib and tepotinib. Monoclonal antibody therapy is divided into anti-MET antibodies, such as onartuzumab and emibetu- zumab, and anti-HGF antibodies, including ficlatuzumab and rilotumumab [12].

As MET is a potential therapeutic target, information regarding its prevalence and clinicopathological relevance is important. However, the prevalence of MET overexpression and gene amplification in GBCs and its clinicopathological significance is not yet known precisely. Therefore, this study evaluated the expression level and gene copy number of MET in GBC tissues and their correlations with various clin-

icopathological characteristics, including patient’s prognosis.

Materials and Methods

1. Patients and specimens

We examined 116 GBC samples (113 patients) obtained from consecutive surgical procedures performed at the Hanyang University Hospital between 1991 and 2016. Three patients had synchronous tumors in the gallbladder. The GBCs were assessed according to the system for staging primary tumor/regional lymph nodes/distant metastasis (TNM) described in the 8th American Joint Committee on Cancer (AJCC) cancer staging manual and histological classification according to the World Health Organization classification of tumors. The clinical data were retrospectively obtained from electronic medical records and included age at diagnosis, sex, pathological profiles (size, stage, histological grade, location, margin status, lymphovascular invasion, and perineural invasion), metastasis, recurrence, and date of death. Tumor recurrence was defined as tumor growth in any site of the body after the surgery, which was diagnosed clinically, radiologically, or pathologically, but mainly by computed tomography and ultrasonography. Among the 113 patients, 44 (37.9%) were men, and the median age was 63.5 years (range, 28 to 90 years). The tumor types of gallbladder were as follows: adenocarcinoma (n=109), squamous carcinoma (n=2), adenosquamous carcinoma (n=4), and small cell carcinoma (n=1). The detailed clinicopathological features are described in Table 1.

2. Tissue microarray construction

Hematoxylin and eosin slides made from formalin-fixed paraffin-embedded tissue blocks were reviewed, and the most representative tumor regions without necrosis and hemorrhage were carefully selected and marked. Single tissue cores, each 3.0 mm in diameter, were punched from each paraffin block and assembled into a new recipient paraffin block using a manual tissue microarray (TMA) instrument (Quick-Ray, Unitma, Seoul, Korea). A total of four TMA blocks were constructed.

3. Immunohistochemistry

Immunohistochemical staining (IHC) was performed on Ventana BenchMark XT autostainer (Ventana Medical Sys- tems, Tucson, AZ), according to the manufacturer’s protocol.

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The primary antibody used was anti-MET (clone sp44) rabbit monoclonal antibody (Ventana Medical Systems).

Two pathologists (K.J. and Y.K.) independently evaluated MET IHC. The immunoreactivity was scored using a four-

tier system, which has been established in previous studies on MET IHC scoring system in NSCLC [13]. This system was used to evaluate both proportion and staining intensity: 0, no staining or < 50% tumor cells with any intensity; 1+, ! 50%

of tumor cells staining with weak or higher staining but

< 50% with moderate or higher strong intensity; 2+, ! 50% of tumor cells with moderate or higher staining but < 50% with strong intensity; 3+, ! 50% of tumor cells staining with strong intensity. Samples exhibiting 2+ or 3+ immunostaining were considered positive for MET overexpression.

4. In situhybridization

MET silver in situhybridization (SISH) was performed on an automated Ventana BenchMark XT platform (Ventana Medical Systems), according to the manufacturer’s protocols and using both MET-specific and centromere 7 (CEP7)-specific probes. Fluorescence in situhybridization (FISH) was used to confirm MET-amplified cases by SISH. FISH was performed using Zytolight SPEC MET/CEN7 Dual color probe (Zytovision, Bremerhaven, Germany), according to the manufacturer’s protocol.

The gene copy number was assessed independently by two pathologists (K.J. and Y.K.) and the number of MET and CEP7 signals was counted in at least 40 tumor cell nuclei in

“hot spot” area (80 nuclei if there was no agreement). Then, the mean number of MET and CEP7 copies per nuclei and the MET/CEP7 ratio was calculated. We used the four different scoring systems for in situhybridization (ISH) interpretation.

- Method A: High-level amplification was defined in tumors with a MET/CEP7 ratio ! 2.0, an average MET gene copy number per cell of ! 6.0, or ! 10% of tumor cells containing ! 15 MET signals. Intermediate level of gene copy number gain being defined as ! 50% of cells containing ! 5 MET signals and criteria for high-level amplification are not fulfilled. Low level of gene copy number gain was defined as ! 40% of tumor cells showing ! 4 MET signals and criteria for high-level amplification or intermediate level of gene copy number gain are not fulfilled. All other tumors were classified as negative [14].

- Method B: Tumors with a mean gene copy number of ! 5 MET signals per tumor cell were classified as METamplification [15].

- Method C: Tumors with a MET/CEP7 ratio ! 2 were defined as METamplification by PathVysion [16,17].

- Method D: High-level amplification (presence of loose or tight clusters of MET signals too numerous to count) was defined in tumors with MET/CEP7 ratio more than 5.0.

Low-level amplification was defined in tumors with MET/CEP7 ratio ! 2.2 and " 5.0 [18].

Table 1. Histological and clinical characteristics of gallbladder cancer patients

AJCC, American Joint Committee on Cancer.

Clinicopathological characteristic No. (%) Age (yr)

< 65 60 (53.1)

! 65 53 (46.9)

Sex

Male 44 (38.9)

Female 69 (61.1)

Histological type

Adenocarcinoma 109 (94.0)

Squamous 2 (1.7)

Adenosquamous 4 (3.4)

Small cell neuroendocrine 1 (0.9)

Histological grade

In situ 6 (5.5)

Well 26 (23.9)

Moderate 50 (45.9)

Poor 27 (24.8)

T category

Tis 6 (5.3)

T1a 8 (7.1)

T1b 8 (7.1)

T2 46 (40.7)

T3 37 (32.7)

T4 8 (7.1)

AJCC stage

0 6 (5.3)

# 16 (14.2)

$ 31 (27.4)

% 44 (38.9)

& 16 (14.2)

Lymph node metastasis

N0 56 (57.1)

N1 31 (31.6)

N2 11 (11.2)

Distant metastasis

M0 103 (91.2)

M1 10 (8.8)

Lympho-vascular invasion

Absent 58 (64.3)

Present 55 (35.7)

Perineural invasion

Absent 72 (63.7)

Present 41 (36.3)

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5. Targeted sequencing by Oncomine comprehensive assay

DNA and total RNA were extracted by using RecoverAll Multi-Sample RNA/DNA Isolation Workflow (Invitrogen, Waltham, MA) according to the manufacturer's instructions.

Library preparation for each sample was performed using the multiplex PCR-based Ion Torrent AmpliSeq technology (Thermo Fisher Scientific, Waltham, MA), together with the Oncomine comprehensive assay (OCA) v1 panel (Thermo Fisher Scientific) according to the manufacturer's protocol.

OCA v1 panel covers multiple exons in 143 genes with 156 amplicons. Next generation sequencing (NGS) was performed on the Ion Torrent S5XL using an Ion 540 chip (PGM, Life Technologies, Grand Island, NE). Results were analyzed using the Variant caller by aligning the reads to the hg19 reference genome, calling the variants, and generating an inter- active report for visualization and quality control. Data analysis was performed using Ion Reporter Server hosting informatic tools (Ion Reporter Software v5.0) for variant analysis, filtering, and annotations.

6. Statistical analysis

To analyze the association between the clinicopathological features and METgene copy number status as well as MET protein expression, chi-square test was employed. Disease- free survival and overall survival (OS) were determined using Kaplan-Meier survival curves, and the log-rank test was used to compare the differences. The Cox proportional hazard regression model was used to evaluate the independent prognostic significance. All statistical analyses were car- ried out using SPSS for Windows ver. 19.0 software (SPSS Inc., Chicago, IL), and p < 0.05 was considered as statistically significant.

7. Ethical statement

This study was approved by the Institutional Review Board of Hanyang University Hospital (IRB No. 2018-08-031- 002). Waiver of informed consent for this study was obtained from IRB based on the retrospective analyses of archived tissues and clinical data.

Results

1. MET overexpression and gene amplification in GBCs

MET IHC was successfully performed in 93/116 tissue cores (80.2%). The unsuccessful staining was mainly due to empty TMA cores or absence of tumor cells. Many tissue cores showed focal or diffuse cytoplasmic/membranous staining pattern with varying intensity. MET IHC score 0-3 was observed in 26 (28.0%), 30 (32.3%), 22 (23.7%), and 15 (16.1%) tumors, respectively. MET overexpression, defined by IHC score 2 or more, was found in 37/93 (39.8%) GBC tumor tissues (Fig. 1).

SISH was successful in 93/116 tissue cores (80.2%), and 17/93 (18.3%), 9 (9.7%), 12 (12.9%), and 11 (13.4%) showed METgene amplification according to four different interpretation methods (Table 2). FISH was performed in same TMA sections and all cases showed concordant results with SISH.

The representative images of SISH and FISH are shown in Fig. 2.

To identify the intratumoral heterogeneity of METcopy number, we performed ISH on whole tissue sections in subset of amplified cases (6 out of 17 cases). Because the assess- ment of heterogeneity is difficult on FISH slide due to dark field, we performed bright field SISH to observe morpho-

Fig. 1. Representative sections of immunohistochemical (IHC) scores for MET expression in gallbladder cancers (!400). (A) IHC score 3, strong cytoplasmic/membranous staining in almost all tumor cells. (B) IHC score 2, more than 50% of tumor cells with moderate staining intensity but < 50% strong intensity. (C) IHC score 1, weak staining in more than 50% of tumor cells. (D) IHC score 0, no staining observed in invasive tumor cells.

A B C D

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logic detail. As we expected, intratumor heterogeneity of MET copy number was found within the tumors (Fig. 3).

Tumors with high-level MET amplification tended to express higher MET protein expression than those with low-

level copy number gain or no copy number gain (S1 Table).

METgene copy number was marginally higher for MET overexpressed tumors. (mean METgene copy number, 2.2 vs. 1.9). However, no statistically significant correlation was Fig. 2. Representative sections of silver in situhybridization (SISH) and fluorescence in situhybridization (FISH) analyses of MET/centromere 7 (CEP7) (!1,000). (A) SISH, no METamplification, 1-2 METgene signals (black), and 1-2 CEP7 signals (red) were present in each nucleus. (B) SISH, low-grade METcopy number gain, 2-7 METgene signals (black), and 1-2 CEP7 (red) were present in each nucleus. (C) SISH, high-level amplification, 7-8 or more METgene signals (black), and 1-5 CEP7 (red) were present in each nucleus. (D) FISH, high-level amplification, 5-10 or more METgene signals (green), and 4-5 CEP7 (red) were present in each nucleus.

A B C D

Table 2. Prevalence of METamplification in gallbladder cancers according to different interpretation criteria

Reference Classification No. No. of amplified cases (%)

Method A Schildhaus et al. [14] High 12 17 (18.3)

Intermediate 0

Low 5

Negative 76

Method B Cappuzzo et al. [15] Positive 9 9 (9.7)

Negative 84

Method C PathVision [16,17] Positive 12 12 (12.9)

Negative 81

Method D Ou et al. [18] High 0 11 (13.4)

Low 11

Negative 82

Fig. 3. Representative section of silver in situhybridization on whole section. (A) Yellow and red circle highlight the non- amplified lesion and amplified lesion, respectively. (B) The tumor cells exhibit 1-2 c-METgene signals (black), and 1-2 centromere 7 signals (red) in each nucleus. (C) In the amplified lesion, tumor cells exhibit 4-12 c-METgene signals (!1,000).

A B C

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CharacteristicNo.Method AMethod BMethod CMethod D NegativePositivep-valueNegativePositivep-valueNegativePositivep-valueNegativePositivep-value Age (yr) < 65 4740 (85.1)7 (14.9)0.43242 (89.4)5 (10.6)> 0.9941 (87.2)6 (12.8)> 0.9942 (89.4)5 (10.6)0.759 ! 654636 (78.3)10 (21.7)42 (91.3)4 (8.7)40 (87.0)6 (13.0)40 (87.0)6 (13.0) Sex Male3630 (83.3)6 (16.7)0.79131 (86.1)5 (13.9)0.30131 (86.1)5 (13.9)> 0.9931 (86.1)5 (13.9)0.745 Female5746 (80.7)11 (19.3)53 (93.0)4 (7.0)50 (87.7)7 (12.3)51 (89.5)6 (10.5) Histological grade G1, G26960 (87.0)9 (13.0)0.03866 (95.7)3 (4.3)0.01164 (92.8)5 (7.2)0.01165 (94.2)4 (5.8)0.006 G31912 (63.2)7 (36.8)14 (73.7)5 (26.3)13 (68.4)6 (31.6)13 (68.4)6 (31.6) T category Tis, T1, T26156 (91.8)5 (8.2)0.00159 (96.7)2 (3.3)0.00758 (95.1)3 (4.9)0.00358 (95.1)3 (4.9)0.007 T3, T43220 (62.5)12 (37.5)25 (78.1)7 (21.9)23 (71.9)9 (28.1)24 (75.0)8 (25.0) N category N04641 (89.1)5 (10.9)0.01145 (97.8)1 (2.2)0.00344 (95.7)2 (4.3)0.00344 (95.7)2 (4.3)0.006 N1, N23321 (63.6)12 (36.4)25 (75.8)8 (24.2)23 (69.7)10 (30.3)24 (72.7)9 (27.3) M category M08669 (80.2)17 (19.8)0.34277 (89.5)9 (10.5)> 0.9974 (86.0)12 (14.0)0.36775 (87.2)11 (12.8)0.593 M177 (100)0 (7 (100)0 (7 (100)0 (7 (100)0 ( AJCC stage I, II4846 (95.8)2 (4.2)< 0.00147 (97.9)1 (2.1)0.01347 (97.9)1 (2.1)0.00147 (97.9)1 (2.1)0.003 III, IV4530 (66.7)15 (33.3)37 (82.2)8 (17.8)34 (75.6)11 (24.4)35 (77.8)10 (22.2) Lymphovascular invasion Absent4943 (87.8)6 (12.2)0.18146 (93.9)3 (6.1)0.46645 (91.8)4 (8.2)0.33646 (93.9)3 (6.1)0.180 Present4333 (76.7)10 (23.3)38 (88.4)5 (11.6)36 (83.7)7 (16.3)36 (83.7)7 (16.3) Perineural invasion Absent5850 (86.2)8 (13.8)0.16155 (94.8)3 (5.2)0.06754 (93.1)4 (6.9)0.02655 (94.8)3 (5.2)0.015 Present3324 (72.7)9 (27.3)27 (81.8)6 (18.2)25 (75.8)8 (24.2)25 (75.8)8 (24.2) Values are presented as number (%). AJCC, American Joint Committee on Cancer.

Table 3.Correlations betweenMETamplification and various clinicopathological parameters of gallbladder cancer patients

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found between MET protein overexpression and gene copy number changes.

Targeted sequencing data was available in 17 cases. MET copy number ranged from 0 to 9.9 (mean, 2.53). No MET- amplified case was detected by NGS method in all 17 cases (S2 Table).

2. Correlation between MET overexpression/amplification and clinicopathological characteristics in GBCs

MET protein expression by immunohistochemistry, including the proportion of positive tumor cells, the staining intensity, and IHC scores, were not correlated with any clinicopathological characteristics (S3 Table).

METamplification by the criteria of Schildhaus et al. [14]

(method A) significantly correlated with higher histological grade (p=0.038), advanced T category (p=0.001), frequent lymph node metastasis (p=0.011), and advanced AJCC stage (p < 0.001). MET-amplified tumors by the criteria of Cap- puzzo et al. [15] (method B) showed higher histological grade (p=0.011), advanced T category (p=0.007), frequent lymph node metastasis (p=0.003), and advanced AJCC stage (p=0.013).

MET amplification by the criteria of PathVysion [16,17]

(method C) correlated with higher histological grade (p=0.011), advanced T category (p=0.003), frequent lymph node metastasis (p=0.003), presence of perineural invasion (p=0.026), and advanced AJCC stage (p=0.001). METamplification by the criteria of Ou et al. [18] (method D) correlated with higher histological grade (p=0.006), advanced T category (p=0.007), frequent lymph node metastasis (p=0.006), presence of per- Fig. 4. Kaplan-Meier survival curves of gallbladder cancer patients stratified on the basis of METamplification status. (A) Recurrence-free survival (RFS) according to METamplification (log-rank test, p=0.436). (B) Overall survival (OS) according to METamplification (log-rank test, p=0.121).

RFS

0 60 80

20

0 100 Time (mo)200 300 400 100

40

A

p=0.436

No c-MET amplification c-MET amplification

OS

0 60 80

20

0 100 Time (mo)200 300 400 100

40

B

p=0.121

No c-MET amplification c-MET amplification

Variable Recurrence-free survival Overall survival

HR 95% CI p-value HR 95% CI p-value

Age (< 65 yr vs. ! 65 yr) 1.075 0.687-1.682 0.750 1.388 0.870-2.214 0.169

Sex (male vs. female) 1.324 0.830-2.112 0.239 1.206 0.745-1.953 0.447

Histologic grade (G1, G2 vs. G3) 1.698 0.923-3.122 0.088 0.813 1.263-2.604 0.001

T category (Tis, T1, T2 vs. T3, T4) 2.464 1.565-3.880 < 0.001 2.539 1.575-4.093 < 0.001 Lymph node metastasis (N0 vs. N1, N2) 2.462 1.470-4.124 0.001 2.749 1.584-4.769 < 0.001 Distant metastasis (M0 vs. M1) 5.639 2.733-11.639 < 0.001 6.157 2.984-12.703 < 0.001

AJCC stage (I, II vs. III, IV) 2.705 1.681-4.353 < 0.001 2.870 1.731-4.759 < 0.001

Lymphovascular invasion (absent vs. present) 2.457 1.545-3.909 < 0.001 2.753 1.698-4.464 < 0.001 Perineural invasion (absent vs. present) 3.045 1.856-4.994 < 0.001 3.643 2.185-6.071 < 0.001 METamplification (absent vs. present)^a) 1.299 0.668-2.525 0.299 1.667 0.868-3.202 0.125 MET overexpression (score 0-1 vs. score 2-3) 0.935 0.587-1.490 0.777 1.040 0.641-1.688 0.873 AJCC, American Joint Committee on Cancer. ^a)By the criteria of Schildhaus et al. [14] (method A).

Table 4. Univariate Cox regression analysis of variables related to the prognosis of gallbladder cancer

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ineural invasion (p=0.015), and advanced AJCC stage (p=0.003) (Table 3).

3. Survival analysis of MET expression and gene amplifi- cation in GBCs

The median recurrence-free survival (RFS) and OS for all patients were 38.6 months and 64.7 months, respectively.

Survival analyses were performed to evaluate the conven- tional pathological prognostic factors of our cohort. Histo- logical grade (p=0.088 and p=0.001), advanced T stage (both p < 0.001), lymph node metastasis (p=0.001 and p < 0.001), distant metastasis (both p < 0.001), advanced AJCC stage (both p < 0.001), lymphovascular invasion (both p < 0.001), and perineural invasion (both p < 0.001) were revealed as predictors of poor RFS and OS, respectively (Table 4).

Neither MET protein expression nor METamplification status showed a significant association with patient’s survival (Table 4, Fig. 4). However, patients with MET-amplified tumor, defined by criteria of Schildhaus et al. [14] (method A), exhibited shorter OS compared to those with the non- amplified tumor (median OS, 26.8 months vs. 72.3 months).

The median RFS of the patients with MET-amplified tumor was also shorter than those with the non-amplified tumor (median RFS, 16.0 months vs. 51.7 months). Similar results were observed with other criteria for METamplification.

Discussion

MET is a transmembrane protein, which acts as a receptor of HGF. The binding of HGF to the MET protein triggers several downstream signaling pathways, including PI3K/AKT, MAPK, Wnt/!-catenin, and STAT signaling pathways [19].

Activation of these pathways can drive various biological processes related to cancer, such as cell proliferation, migra- tion, invasion, inhibition of apoptosis, epithelial to mesenchymal transition, and angiogenesis [8,19]. MET was first identified in a human osteosarcoma as an oncogene, in the form of TPR-MET fusion [20]. Then, dysregulation of MET protein and amplification had been found in several human malignancies including gastric [21], colorectal [22], breast [23], and NSCLC [10].

The oncogenic role of MET was also investigated in biliary tumors, including intra- and extrahepatic cholangiocarcinomas and GBCs. MET overexpression has been observed in the neoplastic glandular epithelium of furan-induced rat cholangiocarcinomas [24]. Previous studies with clinical samples have investigated MET expression by IHC and demon- strated that the prevalence of MET overexpression in GBCs StudyNo. of patientsDetectionCut-off for METPrevalence of METClinicopathological overexpressionoverexpression (%)correlation Moon et al. [28]28 Invasive GBCIHCStaining intensity with 1+, 2+ or74 of GBCs, especially in theAssociated with depth 8 In situcarcinoma3+, more than 10% of tumor cellsinvasive frontof invasion 7 Adenoma Nakazawa et al. [25]89 GBCIHCStaining intensity with 2+ or 3+5.6No correlation FISHNo METamplification detected Sanada et al. [27]14 Biliary cancers, IHCDiffuse membranous or28.6 of biliary cancersN/A including 5 GBCscytoplasmic staining in greater than 30% Yang et al. [26]108 GBCsIHCPercentage of positive tumor cells55.6Associated with differentiation, with 25% or morelymph node metastasis, depth of invasion, poor overall survival Heo et al. [29]12 GBCIHCStaining intensity with 2+ or 3+, 41.7No correlation more than 10% of tumor cells GBC, gallbladder cancer; IHC, immunohistochemistry; FISH, fluorescencein situhybridization; N/A, not available.

Table 5.Prevalence of MET overexpression and amplification in previous studies

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was variable and ranged from 5% to 74% (Table 5) [25-29].

This variability might be due to the difference in primary antibodies used in IHC and interpretation criteria for overexpression. Moon et al. [28] revealed that MET overexpression was observed in 74% of the GBC cases and associated with invasion depth and increased staining intensity of MET at the invasive front of GBCs, which suggested their important role in tumor invasion [28]. However, Sanada et al. [27]

found diffuse membranous staining of MET in the intraduc- tal neoplastic components and no staining in the invasive components, suggesting that MET overexpression might be related to early events of carcinogenesis rather than tumor progression [27]. Yang et al. [26] found that MET overexpression correlated with adverse clinicopathological factors, such as lymph node metastasis, invasion, and decreased OS. In our study, MET overexpression was observed in 39.8% of GBCs. The interpretation criteria of overexpression used in this study was adopted from a previous clinical trial, which revealed that dual MET/EGFR inhibition was clinically ben- eficial in NSCLC only in the MET overexpression group.

However, no significant correlation was observed between MET overexpression and clinicopathological features in our GBC cohort. We performed IHC and SISH on whole tissue sections in subset of cases. Although the different MET immunoreactivity and gene copy number is observed at different areas within tumor, there is no tendency of increased MET immunoreactivity or gene copy number at invasive front as compared to tumor center. Although some previous studies have found no clinicopathological significance of MET overexpression [25,29], other studies have shown significant correlation with worse prognostic factors [26,28].

These discrepancies might be due to different ethnicities of cohorts, the definition of overexpression used, and relatively small number of tumor samples.

Only one ISH study on the METgene copy number in GBCs was found in the literature, and it revealed that no amplification was detected in 89 cases of GBCs [25]. The previous studies using NGS data reported that METamplification was rare event in GBCs [30,31]. In our study, no MET amplification was detected by NGS method in 17 available cases. However, we found that 18.3% of GBCs showed MET amplification by ISH. This discrepancy between ISH and NGS methods for METcopy number evaluation may be resulted from intratumoral heterogeneity, contamination of non-neoplastic stromal and immune cells, and different threshold for detecting copy number alteration in high throughput data. However, the most important strength of tissue section based ISH method is that we can discriminate tumor cells from other non-neoplastic components. The tumor cellularity of GBC used in sequencing is generally low and usually less than 50%. In the previous study, there have been reported intratumoral heterogeneity of MET expression

in solid tumors [32,33]. We also identified intratumoral heterogeneity of MET expression and amplification on whole tissue sections.

METamplification, regardless of interpretation criteria, showed significant association with the adverse clinicopathological features, including higher histological grade, advanced pT category, frequent lymph node metastasis, and advanced AJCC stage. Moreover, METamplification showed a tendency to shorten OS and RFS, which were not statistically significant.

We investigated the correlation between MET protein expression and gene amplification. However, there was no correlation between MET protein overexpression and amplification, although the incidence of MET overexpression tended to be higher in tumors from patients with METamplification than that in tumors from patients with no amplification. Some studies have shown lack of correlation between MET overexpression and gene amplification in HCC and NSCLC [34,35]. These studies and our results suggested that other than gene amplification, there are different mechanisms of protein overexpression. It can be inferred that the other mechanisms, including autocrine and paracrine HGF, increased expression of HGF activator, ligand-independent interactions with other receptors, interactions with other active cell-surface receptors and/or epigenetic regulation might play an important role in MET expression without genomic amplification [19,36,37]. Additionally, activation of other oncogenic factors, inactivation of tumor suppressors, such as TP53, and upregulated microRNAs have been known to enhance MET transcription [38].

In conclusion, the present study found that METamplification was observed in approximately 20% of the GBCs, and that it was associated with adverse clinicopathological characteristics, such as histological grade, T category, lymph node metastasis, and AJCC stage. Further studies are needed to elucidate the significance of METamplification as a potential therapeutic target and predictive biomarker for anti- MET-targeted therapy in GBC patients.

Electronic Supplementary Material

Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).

Conflicts of Interest

Conflict of interest relevant to this article was not reported.

Acknowledgments

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of

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Science, ICT & Future Planning) (NRF-2015R1C1A1A01056091) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education

(NRF-2018R1D1A1B07048798). We would like to thank Sungwoong kim, Jeongyun Eom, and Jisook Kim (Department of Pathology, Hanyang University Hospital) for excellent technical assistance.

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